Received: May 2017, Accepted: July 2017 Background: Fatigue in the workers reduces the individual's resistance and eventually leads to the lower performance, increased errors and accidents. This study assessed the fatigue, mental workload and the correlation between them, in operating room personnel of Hamadan hospitals, Iran. Materials and Methods: In a descriptive study, the mental workload and its association with fatigue in operating room personnel were investigated. Total of 188 cases including surgeons, nurses, operating room and anesthesia technicians participated in the study. Data were collected using demographic, fatigue and mental workload questionnaires, and were analyzed using SPSS software. Results: The mean of overall mental workload was 74.3 ± 10.8. The results also showed that overall fatigue and its domains, including subjective fatigue, concentration, motivation and physical activity were 48.73%, 54.31%, 51.65%, 38.96% and 42.06%, respectively. Among different domains of mental workload, performance (r = -0.380, P < 0.001) and frustration (r = 0.222, P = 0.014) were significantly associated with overall fatigue. Between overall mental workload and overall fatigue, there was not a significant correlation (r = 0.029, P = 0.749). Among different dimensions of fatigue, only subjective fatigue was correlated with mental workload (r = 0.255, P = 0. 005). Conclusions: Regarding the association of performance and the frustration with overall fatigue and the association of overall mental workload with subjective fatigue, it can be concluded that mental workload and fatigue may be correlated with each other. Therefore, any reduction in mental workload domains may lead to fatigue reduction in the operating room personnel.